CN105845539B - Fragment ion mass spectrum is measured using time-of-flight mass spectrometry instrument - Google Patents
Fragment ion mass spectrum is measured using time-of-flight mass spectrometry instrument Download PDFInfo
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- CN105845539B CN105845539B CN201610076521.4A CN201610076521A CN105845539B CN 105845539 B CN105845539 B CN 105845539B CN 201610076521 A CN201610076521 A CN 201610076521A CN 105845539 B CN105845539 B CN 105845539B
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/26—Mass spectrometers or separator tubes
- H01J49/34—Dynamic spectrometers
- H01J49/40—Time-of-flight spectrometers
- H01J49/405—Time-of-flight spectrometers characterised by the reflectron, e.g. curved field, electrode shapes
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/004—Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/26—Mass spectrometers or separator tubes
- H01J49/34—Dynamic spectrometers
- H01J49/40—Time-of-flight spectrometers
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/0027—Methods for using particle spectrometers
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/0027—Methods for using particle spectrometers
- H01J49/0036—Step by step routines describing the handling of the data generated during a measurement
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/004—Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn
- H01J49/0045—Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn characterised by the fragmentation or other specific reaction
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Abstract
The mass spectrographic method of fragment ion is obtained using time of-flight mass spectrometer the present invention provides one kind, wherein obtaining the mixing mass spectrum of the fragment ion of different precursor ion species and being compared to each other, so that it is determined that the signal of the fragment ion from identical precursor ion species.The time of-flight mass spectrometer includes ion gun, flight path, reflector and ion detector.Flight path is preferably field-free and is located at before reflector, and reflector is preferably provided with the increased reflection potential of quadratic power.
Description
Technical field
The present invention relates to the mass spectrum using time-of-flight mass spectrometry instrument measurement fragment ion.
Background technology
Note：Use " dalton " (Da) unit herein, and unofficial " unified atomic mass unit " (u), Da units
" International System of Units (the SI) " file of upper one edition (the 8th edition) of " International Bureau of Wieghts and Measurements " it has been added in 2006, with atomic mass
Unit is in par.As described in this document, so do primarily to allowing to use the class such as kilodalton and milli dalton
Like unit.
Mass spectrograph is typically only capable to determine the ratio of mass of ion and ionic charge.Hereinafter, term " quality of ion "
Or " mass of ion " also refers to the ratio of the quality m and the unnecessary positive or negative elementary charge quantity z of ion of ion, i.e. quality-elementary charge ratio
(or abbreviation mass-to-charge ratio) m/z.
102013011462.4 (C. of patent document DE herein will be incorporated by reference) in, retouch
State with one or more Cassini reflectors and the time of-flight mass spectrometer of non-generic Mamyrin reflectors.In the disclosure
In also the prior art is described in detail.
Term " fragment ion mass spectrum " or " daughter ion mass spectrum " are often referred to the mass spectrum of the fragment ion of selected ionic species, and
It is commonly referred to as " parent ion " to be selected to broken ionic species.
In the time of-flight mass spectrometer of substance assistant laser desorpted ionized (MALDI), for producing two kinds of daughter ion
It is distinct between broken type (ISD crushes (" decaying in source ") and PSD is crushed (" being decomposed behind source ")).In order to pass through PSD side
Formula obtains daughter ion mass spectrum, can will increase to such degree for the energy of the laser pulse of MALDI during MALDI,
Produce many metastable state analyte ions and these ions decayed to only after the first accelerating region and before reflector it is broken
Piece ion (daughter ion).It can also be produced by way of being collided in the inflation collision cell between the first accelerating region and reflector
Raw unstable parent ion.The parent ion for obtaining daughter ion mass spectrum must be selected in both cases.Usually using female
Ion selector selects parent ion, and the selection device be located at the first accelerating region afterwards and before reflector, if collision cell, also position
Before collision cell.If metastable state parent ion decomposes between the first accelerating region and parent ion selector, here
The fragment ion formed may also pass through parent ion selector, this is because they have substantially with undecomposed parent ion
Identical speed, therefore parent ion selector is reached at the same time with undecomposed parent ion.Undecomposed parent ion and by selected mother
The daughter ion that ion is formed usually initially passes through the second accelerating region, is then separated in reflector, and be measured as daughter ion
Mass spectrum.For example, in 198 56 014C2 (C. of patent document DEEt al., corresponding to GB 2 344 454B and US 6,
Time of-flight mass spectrometer is described in 300,627B1) and obtains the correlation method of PSD daughter ion mass spectrums.
Relatively large sample material will be consumed by obtaining daughter ion mass spectrum by substance assistant laser desorpted ionized (MALDI)
Amount.For PSD is crushed, (thus also increasing sample consumption at the same time) is significantly increased in the energy of laser pulse, with produce by
The a large amount of metastable state ions decomposed in the first straight line flight path before reflector.In addition, it can also be directed to each selected female
Ionic species obtains single daughter ion mass spectrum.It is clear that this there are a kind of shortcoming, that is, it is a kind of to be only limitted to processing every time
Precursor ion species, this means that a lot of other ionic species are filtered and are not used by, therefore, if to measure multiple
Daughter ion mass spectrum, then can consume relatively large sample size.Another disadvantage is that, it is necessary to multiple daughter ion mass spectrums are sequentially obtained,
This just needs longer time of measuring.
The content of the invention
The object of the present invention is to provide the quick flight time matter for obtaining fragment ion in the case of consumption sample on a small quantity
The method of spectrum, is realized particular by substance assistant laser desorpted ionized (MALDI).
The present invention is provided using the time of-flight mass spectrometer measurement mass spectrographic method of fragment ion, the time of-flight mass spectrometer bag
Containing ion gun, flight path, reflector and ion detector.
First method according to the present invention is characterized in that：(a) at least two are obtained using different instrument parameters every time to mix
Time of flight spectrum is closed, wherein mixing time of flight spectrum includes the signal of the fragment ion of more than one precursor ion species, and it is broken
Piece ion produces in the flight path before reflector；(b1) is compared to each other mixing time of flight spectrum, and identifies and come from one
The signal of the fragment ion of a precursor ion species；And/or (b2) determines a fragment ion species in time of flight spectrum is mixed
Flight time, and thus calculate the mass-to-charge ratio of fragment ion and related parent ion.If the electric charge of fragment ion and parent ion
It is known that it can then determine " real " quality.The instrument parameter being modified can be, for example, accelerating region before flight path
Accelerating potential, either the decelerating voltage at reflector or the two.
If one or more mixing time of flight spectrum also includes the signal of parent ion, when flight can be mixed by comparing
Between spectrum the signal of the fragment ion from a precursor ion species is assigned to corresponding precursor ion species.It will can have determined
The signal of fragment ion from precursor ion species letter with the precursor ion species with the signal of the precursor ion species or not
Number together it is changed into " separated " pure fragment ion mass spectrum.
Identical fragment ion species can be derived from by selection one in each spectrum in two mixing time of flight spectrum
Signal, and be determined as the mass-to-charge ratio of the solution of following equalities, the mass-to-charge ratio of fragment ion and related parent ion calculated with this：
T1=Sys (m/qm,M/qM,P1) (1)
T2=Sys (m/qm,M/qM,P2) (2)
Wherein, M and qMIt is the quality and electric charge of parent ion respectively；M and qmIt is the quality and electric charge of fragment ion respectively；T1
And T2It is the flight time of two signals of the fragment ion species determined according to mixing time of flight spectrum；P1And P2It is to be modified
Be used for obtain time of flight spectrum instrument parameter value；And Sys is the system function of time of-flight mass spectrometer, wherein fragment
The flight time of ion is the function of instrument parameter and fragment ion to the mass-to-charge ratio of related parent ion.
Calculating fragment ion is additionally may included in each in several mixing time of flight spectrum to related mother ion mass-to-charge ratio
One is selected in a spectrum and is derived from the signal of identical fragment ion species, and is determined as regression equation Ti=Sys (m/qm,M/qM,
Pi) parameter mass-to-charge ratio：Wherein, M and qMIt is the quality and electric charge of parent ion respectively；M and qmIt is the quality of fragment ion respectively
And electric charge；TiIt is the flight time of the signal of the fragment ion species determined according to mixing time of flight spectrum；And PiIt is to be used to obtain
Obtain the value of the instrument parameter of time of flight spectrum.When the system function Sys of time of-flight mass spectrometer gives the flight of fragment ion
Between be the function of instrument parameter and fragment ion to the mass-to-charge ratio of related parent ion.
Second method according to the present invention is characterized in that：(a) a mixing time of flight spectrum is obtained, which includes one
The signal of the fragment ion of above precursor ion species, wherein fragment ion produce in the flight path before reflector；(b) select
Select two signal S in the isotopic pattern of a fragment ion1And S2, their flight time T is determined by mixing flight spectrum1
And T2；And (c) determines mass-to-charge ratio of the fragment ion to related parent ion as the solution of following equalities：
T1=Sys (m/qm,M/qM) (3)
T2=Sys ((m+nDa)/qm,(M+n·Da)/qM) (4)
Wherein, M and qMIt is the quality and electric charge of parent ion respectively；M and qmIt is the quality and electric charge of fragment ion respectively；Institute
Select isotope that there is the of poor quality of n dalton；And Sys is the system function of time of-flight mass spectrometer, wherein fragment ion
Flight time is function of the fragment ion to the mass-to-charge ratio of related parent ion.If the electric charge of fragment ion and related parent ion is
Know, then can determine " real " quality m and M.In the method according to the invention, although when fragment ion generates (especially
When metastable state parent ion decomposes) resolution to be received is on the direction of statistical distribution, but stills need relatively long fly
The row time could parse isotopic pattern.
Can be out of, field-free flight path between such as ion gun and flight path or before fragment ion formation or generation
Large number of ionic species in select several precursor ion species.It need not select single precursor ion species.
Entering flight path preacceleration parent ion, such as accelerating in ion gun itself, or positioned at ion gun and flying
Accelerate in accelerating region between walking along the street footpath.The flight time of ion is usually since acceleration fields are opened in pulse.Can be sub- by decomposing
Stable state parent ion forms fragment ion in flight path, and/or produces fragment ion by parent ion in broken pond.Parent ion
And its fragment ion fly to reflector flight time it is preferably identical, but through reflector flight time it is preferably different.Instead
Emitter be preferably provided with the increased retarding potential of quadratic power, the especially Potential Distributing of Cassini ion traps more will in this way, so as to
In decoupling ion vertical and horizontal vibration.After reflector, ion can be to pass through the second accelerating region or the second flight road
Flight path before footpath, the second accelerating region or the second flight path most can be compared to reflector is short, is then examined in ion detector
Measured ion.
Obtain mixing time of flight spectrum and might mean that the quality of ion signal is no longer along flight time axis monotone increasing,
That is, compared with the fragment ion of other parent ions or the precursor ion species of the second weight, the fragment ion of the first precursor ion species can
There can be the longer flight time, therefore flight time axis cannot be converted directly into quality axis.Pass through the single letter of fragment ion
Number, it is impossible to determine its quality relevant to electric charge or related parent ion and the relevant quality of electric charge.
If total flight path of time of-flight mass spectrometer is made of the field-free flight path before reflector substantially, and such as
Fruit reflector has with the increased retarding potential of quadratic power, then system function is：
Wherein, mp,dAnd qmIt is the quality and electric charge of fragment ion respectively；MpAnd qMBe respectively related parent ion quality and
Electric charge；UBThe accelerating potential of accelerating region before being field-free flight path；And UCIt is the decelerating voltage at reflector.Can be by making
Demarcated with one or more known substances to determine two constant c1And c2。
If use different accelerating potential UB1And UB2Two mixing time of flight spectrum are obtained, then can obtain fragment ion
The following equalities of flight time：
Quadratic power retarding potential combined with field-free flight path it is highly beneficial, this is because what can easily be measured from two is mixed
Close identification in time of flight spectrum and be derived from a kind of fragment ion of precursor ion species.It can be seen that, quality is from the two equatioies
MpAll quality of precursor ion species be mp,dThe fragment ion (and parent ion is in itself) of (wherein d=1,2,3 ...) has phase
Same flight time delay Δ T=T2-T1, this is only dependent upon the mass-to-charge ratio M of parent ionp/qMWith two accelerating potentials：
There are all of the second mixing time of flight spectrum of delay Δ T of identical flight time with the first mixing time of flight spectrum
Signal is identified as being derived from a precursor ion species.The mass M of precursor ion species hereinpCan directly by the flight time delay and
Accelerating potential UB1And UB2Determine.Flight time delay (Δ T (the M of fragment ion from different precursor ion species1),ΔT
(M2) ...) can also be determined by the cross-correlation between the first and second mixing time of flight spectrum.From a precursor ion species
The signal of daughter ion can be identified as in two mixing frequency spectrums in one of the definite flight time delay overlapping signal in place.This
Outside, due to two mix time of flight spectrum in remain precursor ion species daughter ion order, can be derived from it is identical
Two signals S1 and S2 are easily found in the mixing time of flight spectrum of daughter ion species.
It can also use different accelerating potential UB1And UB2Two mixing time of flight spectrum are obtained, mother is also included in the spectrum
The signal of ion.In the second mixing time of flight spectrum, then can identify has identical flight with the first mixing time of flight spectrum
The signal of time delay Δ T.Signal with the most long flight time is appointed as precursor ion species, other signals are appointed as source
From the fragment ion of the precursor ion species.
Except one or more mixes time of flight spectrum, at least one flight time without fragment ion is preferably also obtained
Spectrum, so as to the parent ion that the Direct Recognition in time of flight spectrum is mixed is also undecomposed or broken, so that itself and daughter ion be distinguished.
Can be also U in accelerating potentialB1And UB2When additionally obtain two time of flight spectrum, the signal of parent ion is only included in the spectrum.Then
Second can be mixed in time of flight spectrum and be specified with the signal that the first mixing time of flight spectrum has delay Δ T of identical flight time
For the precursor ion species that there is the identical flight time to postpone Δ T in the time of flight spectrum additionally obtained.
The ion gun that ion is produced by substance assistant laser desorpted ionized (MALDI) is preferred.In this case,
MALDI ion guns are preferably directly bordered by field-free flight path.Then generally axially accelerate the ion from MALDI ion guns, allow
It enters field-free flight path.In MALDI ion guns, sample, therefore sample are bombarded with high pulse energy by laser pulse
Many internal energies are usually absorbed, so that at least a portion ion produced is metastable state, and the flight before reflector
Fragment ion is decomposed into path.In addition, flight time mass spectrum can be also obtained in low pulse energy to produce a small amount of or not produce
Raw metastable state parent ion, which is M only comprising quality1、M2、M3Etc. all ions for being probably parent ion.
Brief description of the drawings
Fig. 1 shows the time of-flight mass spectrometer with MALDI ion guns and Cassini reflectors (20,21,22,23)
Rough schematic view, pass through the mass spectrograph and obtain the mixing flight time with several precursor ion species and multiple fragment ions
Spectrum.
Fig. 2 shows the Cassini reflectors of different designs, and the electric field of Cassini reflectors (20) can be produced using it.
Fig. 3 and Fig. 4 shows two manually mixing time of flight spectrum (1,2), the two spectrums, which include, comes from three parent ions
The signal of species (quality 800,900 and 1000 dalton) and its fragment ion.
Fig. 5, Fig. 6 and Fig. 7 show the superposition of the mixing time of flight spectrum (1,2) from Fig. 3 and Fig. 4, wherein every time will
It is mobile to a certain degree to mix time of flight spectrum (2) so that in two mix time of flight spectrum, one of three precursor ion species
Signal is relative to each other.
Fig. 8 shows the rough schematic view of time of-flight mass spectrometer, which comes from ion gun (30)
Ion beam (31).
Embodiment
The present invention provides the quick method for obtaining daughter ion mass spectrum in the case of consumption sample on a small quantity, wherein, with every
It is secondary to determine mode of which fragment ion from which precursor ion species using mathematics geometrical relationship, use the flight time
Mass spectrograph obtains mixing time of flight spectrum from a large amount of parent ions and fragment ion.This time of-flight mass spectrometer includes ion gun, flies
Walking along the street footpath, reflector and ion detector.Flight path is preferably field-free, and reflector is preferably provided with the increased reflection of quadratic power
Potential.
Fig. 1 shows the rough schematic view of time of-flight mass spectrometer, the mass spectrograph include MALDI ion guns (10,11,
12), field-free flight path (14), Cassini reflectors (20,21,22,23) and ion detector (26).
Have a variety of samples on sample disk (10), every kind of all mixtures containing material, using ultraviolet pulse (12) by
Substance assistant laser desorpted/ionization (MALDI) carrys out ionization material.Inside can be produced using ultraviolet pulse (12) bombardment sample disk
The very high parent ion of energy (is known as " metastable state ion "), field-free flight of at least part parent ion after electrode (11) is accelerated
Fragment ion is decomposed on path.It can accelerate ion in MALDI ion guns under time delay, so as to make phase homogeneity every time
The ion time of amount is gathered in entrance (15) place (by " delay is extracted " aggregation).The part metastable state parent ion edge of different material
Field-free flight path (14) decomposes；The speed of fragment ion and parent ion is about the same, therefore enters Cassini reflectors at the same time
(20、21、22、23).However, parent ion and fragment ion with the different flight time along different tracks (16,17,18,19)
Through Cassini reflectors (20,21,22,23).The relatively low kinetic energy of fragment ion (16,17,18) means that they cannot be as mother
Ion (19) equally penetrates deeply Cassini reflectors, and relatively low quality means that they can be with corresponding faster speed
Through reflector (20,21,22,23).But fragment ion and parent ion are spatially gathered on outlet opening (24).It is female
Ion and fragment ion accelerate to high energy (generally between 10 Hes in a short period of time in accelerating region (25) (diaphragm stacking)
Between 30keV), and the mixing mass spectrum as parent ion and fragment ion in ion detector (26) is measured.
The flight time that parent ion and fragment ion fly to Cassini reflectors (20,21,22,23) is identical, but with difference
Flight time temporal aggregate in Cassini reflectors (20,21,22,23), such ion detector (26) is with regard to measurable bag
" the mixing time of flight spectrum " of parent ion and its fragment ion containing several species.
Shown is the cross section of Cassini reflectors (20,21,22,23).Ion trajectory is located at two internal electrodes
(23) between, because electrode is located at outside plotting planes, therefore it is represented by dashed line.Cassini reflectors herein include shell
Electrode (20), two internal electrodes (23) and two termination equipotential plates (21,22), as described in open DE 102013011462A1.
Cassini reflectors (20,21,22,23) have a Potential Distributing of half Cassini ion trap, and potential is in the axial direction accurately
Increased with quadratic power.Equipotential plate (21,22) has an electrode of curve shape, its shape and the Cassini at the equipotential plate from
The Potential Distributing equipotential surface of sub- trap is identical.Equipotential plate (22) has two holes (15,24) for being used to injecting and casting ion, and
The design of the shapes of Cassini reflectors (20,21,22,23) and injection with casting hole (15,24) position is preferably able to allow matter
Identical ion is measured in Cassini reflectors (20,21,22,23) by odd number laterally half vibration.In Fig. 1, ion exists
Cassini reflectors have passed through 3/2 swaying in (20,21,22,23).More elongated Cassini reflections can also be made
Device, it is deeper which penetrates parabola potential in the vertical.Then every half of lengthwise oscillations, ion have to carry out 5/2,7/2
Or 9/2 swaying, which increase the acceptance of the fragment ion very different to quality m.
The Potential Distributing Ψ (x, y, z) of Cassini ion traps can be, for example, the super logarithm field of following form：
The shape of field can be changed by constant ai, b and B.Uln、UquadAnd UoffIt is potential voltage, ClnAnd CquadIt is constant.
The inner surface of exterior chamber (20) and the outer surface of internal electrode (23) are the normal of equipotential surface Ψ (x, y, z)=this Potential Distributing
Number.In cross-section, equipotential lines forms the approximate Cassini ellipses around internal electrode herein；Two internal electrode (23) productions
Raw second order Cassini is oval；It is oval that n internal electrode produces n ranks Cassini., can there are ion for even number internal electrode
With the embodiment of swaying near the median plane between at least a pair of internal electrodes.Form parameter can be used for setting longitudinal direction to shake
Swing any selected ratio between cycle and swaying cycle.
Cassini reflectors are preferred herein, this is because it has with the increased retarding potential of quadratic power (reflection electricity
Gesture), and can also spatially assemble ion in two transverse directions.In addition, in reflector by the fragment that is decomposed to form from
Son has almost been filtered completely.But it in principle, potential can be used to obtain with the increased any reflector of quadratic power to meet
The optimal single system function of formula (5).But herein it should also be noted that can also be used with Mamyrin reflectors
Time of-flight mass spectrometer does not have quadratic power retarding potential to obtain mixing time of flight spectrum according to the present invention using
During reflector, system function is different from equation (5), therefore the corresponding equation group of solution may be more complicated.
The exterior chamber (20) of Cassini reflectors is difficult manufacture in Fig. 1.Moreover, substantially closed Cassini reflections
The inside of device is also not easy to be evacuated.Fig. 2 shows design difference but the Cassini reflectors with phase same electric field：Herein
Exterior chamber (20) in Fig. 1 is substituted by the stacking with identical diaphragm with holes (122).Cassini illustrated herein is anti-
Emitter is three-dimensional；Cut on reflection direction, and illustrate only half of detector.Diaphragm with holes has Cassini elliptical forms
Inside opening.It is the electrode supply of diaphragm with holes (122) and equipotential plate (120) to maintain the electric field of Cassini ion traps
Produce with the suitable potential of the increased field of quadratic power.Equipotential plate (120) and (121) correspond to the equipotential plate in Fig. 1.Different quality
The fragment ion of m is mobile along track (124), these tracks extend to the different depth of reflector.Deeper prolong on parent ion edge
The track (125) reached in reflector is mobile.The present embodiment has the advantages that several：Reflector is easier to be evacuated, overall ruler
Very little smaller, manufacture is simpler, cost is lower.
Fig. 3 and Fig. 4 shows two mixing time of flight spectrum (1,2), they are the time of-flight mass spectrometers used in Fig. 1
By applying two different accelerating potential U in MALDI ion guns (10,11,12)1And U2And obtain.In two mixing flights
In time spectrum (1,2), with arrow mark parent ion, their quality is 800,900 and 1000 dalton.For each parent ion
Species, all there are three kinds of fragment ions that quality is 100,200 and 300 dalton, is painted using the intensity of related precursor ion species
Make every kind of fragment ion.Abscissa represents the flight time.
The flight time of parent ion and its related fragment ion on straight line field-free flight path (14) is identical, but
Flight time in Cassini reflectors (20,21,22,23) is different.If in two different accelerating potential U1And U2Under obtain
Obtain two mixing time of flight spectrum, then for fragment ion, total flight time t1Mixing time of flight spectrum 1 is produced, it is total to fly
Row time t2 produces mixing time of flight spectrum 2：
Wherein, MpIt is the quality (wherein p=1,2 ...) of parent ion, mdBe related fragment ion quality (wherein d=1,
2nd ...), qMAnd qmIt is its electric charge, U1It is the accelerating potential of parent ion in MALDI ion guns (10,11,12), and UCIt is
The decelerating voltage at Cassini reflectors (20,21,22,23) place.It can be demarcated by using known substance to determine two often
Number c1And c2.For MALDI ions, electric charge qMAnd qmThe electric charge of typically each proton.
If the flight time t1 and t2 of a fragment ion species in two mixing time of flight spectrum (1,2) it is known that
It then can determine that the mass M of related parent ionp：
Thus it can determine that the quality m of fragment iond：
If ion passes through the Cassini in Fig. 1 anti-with relatively low energy (such as energy with only 300 electron volts)
Emitter, then the flight time is longer, thus can produce of a relatively high resolution ratio, it means that although fragment ion is decomposing
When the resolution that receives be on the direction of statistical distribution, but still can be that fragment ion parses isotope line.For example, use Fig. 1
In time of-flight mass spectrometer obtain individually mixing time of flight spectrum, if this it is single mixing time of flight spectrum in parse
Fragment ion species13C signal and12C signal, then can obtain parent ion and the unknown following two equation of fragment ion masses：
Wherein, mp,dAnd qmIt is the quality and electric charge of daughter ion respectively；MpAnd qMIt is the quality and electricity of related parent ion respectively
Lotus；UBIt is the accelerating potential of parent ion in MALDI ion guns；And UCIt is the decelerating voltage at reflector.Can be by using known
Material is demarcated to determine two constant c1And c2.Can be by asking solution of equations to obtain two unknown quality.According to the present invention
This method need good time-of-flight resolution, but different accelerating potentials need not be used to obtain for the second mixing flight time
Spectrum.
Also two mixing time of flight spectrum (1) and (2) can be compared to each other, to identify the fragment for being derived from a precursor ion species
The signal of ion.For example, compare can be geometry comparison, or realized by cross-correlation.In two mixing flights
In time spectrum (1,2), parent ion and all related fragment ions postpone identical time-of-flight values t1-t2, this is because two
Under a accelerating potential, the flight time still phase of parent ion and its fragment ion in Cassini reflectors (20,21,22,23)
Together, the flight time only in rectilinear flight path (14) is different.Now, it can easily distinguish which which fragment ion is derived from
A parent ion, shows that time of flight spectrum (1) and the mixing of each self-dalay in Fig. 4 are mixed in Fig. 3 to fly in Fig. 5, Fig. 6 and Fig. 7
The superposition three times of row time spectrum (2).The mixing time of flight spectrum of Fig. 4 is moved along flight time axis, until with 800 dongles
The parent ion of quality or the position of the parent ion with 900 dalton mass or the parent ion with 1000 dalton mass that
This is opposite.
In Figure 5, quality in time of flight spectrum (1) and (2a) is mixed to overlap for the parent ion of 800 dalton；Meanwhile this
In can also easily pick out, all fragment ions of these parent ions also overlap (dotted arrow), therefore can easily distinguish
Related fragment ion.When comparing mixing time of flight spectrum (1) and (2b) or (1) and (2c) in figure 6 and figure 7 in the same manner,
Also recognizable quality is the fragment ion of the parent ion of 900 and 1000 dalton.
Also it can determine to mix for moving in Fig. 5 to Fig. 7 by the cross-correlation between mixing time of flight spectrum (1) and (2)
Three time delays of time of flight spectrum (2a, 2b, 2c) are closed, this cross-correlation has at each time of three time delays
There is local maximum.It can select to delay those signals that mutually should determine that the time from mixing time of flight spectrum pure broken to obtain
Piece ionic spectrum.
The conventional flight time spectrum of parent ion is preferably first obtained in the case of no fragment ion using low laser energy.
It is all ions that M1, M2, M3 etc. are probably parent ion that the spectrum, which includes quality,.If for two mixing time of flight spectrum
(1) and under two accelerating potentials of (2) two time of flight spectrum of parent ion are obtained, then can recognize that two mixing time of flight spectrum
(1) and the parent ion in (2).Represent that the time delay of respective parent ion and its fragment ion characteristic can also be by the two of parent ion
The cross-correlation of a time of flight spectrum determines.If the time of flight spectrum of parent ion includes each and can be analyzed to fragment ion
Too many parent ion (such as possible parent ion more than 50), may be used traditional parent ion separator to select parent ion
Mass range (for example, mass range is between 1000 and 1500 dalton), the quantity of parent ion is limited to for example every
Only have 15 parent ions in a mixing time of flight spectrum.About four to five mixing time of flight spectrum can be used, by this way
Determine the mass spectrum of all 50 or so daughter ions.Therefore, compared with the method used before, sample consumption reduces by 10 times.
Fig. 8 is the rough schematic view for showing time of-flight mass spectrometer, which is just being accompanied each other using impulse generator (32)
Speed comes from the ion beam (31) of ion gun (30).For clarity, depict on a projection plane herein ion gun (30) and from
Beamlet (31)；But they should be located at the position at a right angle with projection plane, can be by vertical with projection plane to produce
Slit (15) enter Cassini reflectors (20,21,22,23) ribbon ion beam (14).The parent ion of pulsed beams passes through broken
The fragment ion for mixing time of flight spectrum is directly decomposed or be decomposed on field-free flight path (14) here to broken pond (33) simultaneously.
For example, crushing in broken pond (33) can be realized by the photon with enough energy or by being touched in inflation broken pond (33)
Hit realization.
Those skilled in the art can have found to be very easy to develop more correlations based on ion reflections device according to the present invention
Embodiment.This patent protection application will also cover such application gone out from derivative of the present invention.
Claims (24)
1. one kind includes ion using the time of-flight mass spectrometer measurement mass spectrographic method of fragment ion, the time of-flight mass spectrometer
Source, flight path, reflector and ion detector, wherein,
(a) at least two mixing time of flight spectrum are obtained using different accelerating potentials every time, the mixing time of flight spectrum bag
Signal containing the fragment ion from more than one precursor ion species, and fragment ion is in the flight path before reflector
Produce；With
(b1) mixing time of flight spectrum is compared to each other, thus to identify the letter of those fragment ions from a precursor ion species
Number；And/or
(b2) determine the flight time of fragment ion species in mixing time of flight spectrum and be used to calculate fragment ion and correlation mother
The mass-to-charge ratio of ion.
2. the method according to claim 1, wherein, select one in each spectrum in two mixing time of flight spectrum and be derived from
The signal of identical fragment ion species, and the solutions of following equalities is sought to determine the mass-to-charge ratio of fragment ion and related parent ion：
T1=Sys (m/qm, M/qM, P1)
T2=Sys (m/qm, M/qM, P2),
Wherein, M and qMIt is the quality and electric charge of parent ion respectively；M and qmIt is the quality and electric charge of fragment ion respectively；T1And T2It is
The flight time of two signals of the fragment ion species determined according to mixing time of flight spectrum；P1And P2It is to be used to be flown
The value of the accelerating potential of time spectrum；And Sys is the flight time of the system function of time of-flight mass spectrometer, wherein fragment ion
It is the function of the mass-to-charge ratio of instrument parameter and fragment ion and related parent ion.
3. the method according to claim 1, wherein selection one is derived from each spectrum in several mixing time of flight spectrum
The signal of identical fragment ion species, and it is determined as regression equation Ti=Sys (m/qm, M/qM, Pi) parameter fragment ion
With the mass-to-charge ratio of related parent ion, wherein M and qMIt is the quality and electric charge of parent ion respectively；M and qmIt is the matter of fragment ion respectively
Amount and electric charge；TiIt is according to mixing time of flight spectrum and the flight time of the signal of definite fragment ion species；PiIt is to be used to obtain
Obtain the value of the accelerating potential of time of flight spectrum；And Sys is the system function of time of-flight mass spectrometer, wherein fragment ion is winged
The row time is the function of the mass-to-charge ratio of instrument parameter and fragment ion and related parent ion.
4. the method according to claim 1, wherein at least one mixing time of flight spectrum has the extra of parent ion, passes through
Compare mixing time of flight spectrum by the signal of those fragment ions from a precursor ion species be assigned to it is corresponding female from
Subcategory.
5. one kind includes ion using the time of-flight mass spectrometer measurement mass spectrographic method of fragment ion, the time of-flight mass spectrometer
Source, flight path, reflector and ion detector, wherein,
(a) mixing time of flight spectrum is obtained, which includes the signal of the fragment ion of more than one precursor ion species, and fragment
Ion produces in the flight path before reflector,
(b) two signal S in the isotopic pattern of fragment ion are selected1And S2, and it is determined according to mixing time of flight spectrum
Flight time T1And T2, and
(c) by asking the solution of following equalities to calculate the mass-to-charge ratio of fragment ion and related parent ion：
T1=Sys (m/qm, M/qM)
T2=Sys ((m+nDa)/qm, (M+nDa)/qM),
Wherein, M and qMIt is the quality and electric charge of parent ion respectively；M and qmIt is the quality and electric charge of fragment ion respectively；It is selected same
Position element has the of poor quality of n dalton；And Sys is the flight of the system function of time of-flight mass spectrometer, wherein fragment ion
Time is the function of the mass-to-charge ratio of fragment ion and related parent ion.
6. method according to claim 5, wherein total flight path includes field-free flight path and reflector, which has
With the increased retarding potential of quadratic power, therefore system function is provided by following equalities：
<mrow>
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<mo>+</mo>
<msub>
<mi>c</mi>
<mn>2</mn>
</msub>
<mo>&CenterDot;</mo>
<msqrt>
<mfrac>
<msub>
<mi>m</mi>
<mrow>
<mi>p</mi>
<mo>,</mo>
<mi>d</mi>
</mrow>
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<mn>2</mn>
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</mfrac>
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Wherein, MpAnd qMIt is the quality and electric charge of parent ion respectively；mp,dAnd qmIt is the quality and electric charge of fragment ion respectively；UBIt is
The accelerating potential of accelerating region before field-free flight path；UCIt is the decelerating voltage at reflector；c1And c2It is constant；And T
(Mp, mP, d) be fragment ion flight time.
7. method according to claim 6, wherein, gather and compare two mixing flight time with different accelerating potentials
Spectrum, will have in the second mixing time of flight spectrum of delay Δ T of identical flight time compared with the first mixing time of flight spectrum
All signals are determined as being derived from a precursor ion species.
8. method according to claim 7, wherein, in addition only included using the accelerating potential of two mixing time of flight spectrum
Two time of flight spectrum of parent ion signal, and will have delay of identical flight time Δ compared with the first mixing time of flight spectrum
Signal in the second mixing time of flight spectrum of T is appointed as the mother in time of flight spectrum with delay Δ T of identical flight time
Ionic species.
9. method according to claim 6, wherein two mixing time of flight spectrum of different accelerating potential acquisitions are used, described two
A mixing time of flight spectrum also includes the signal of related parent ion, and wherein, determined in the second mixing time of flight spectrum with
First mixing time of flight spectrum compares those signals with delay Δ T of identical flight time, and wherein, will have most long fly
The signal of row time is appointed as precursor ion species, other signals are appointed as the fragment ion from the precursor ion species.
10. method according to claim 7, wherein, by the cross-correlation between the first and second mixing time of flight spectrum come really
Surely it is derived from the flight time delay of the fragment ion of different precursor ion species.
11. method according to claim 8, wherein, by the cross-correlation between the first and second mixing time of flight spectrum come really
Surely it is derived from the flight time delay of the fragment ion of different precursor ion species.
12. method according to claim 9, wherein, by the cross-correlation between the first and second mixing time of flight spectrum come really
Surely it is derived from the flight time delay of the fragment ion of different precursor ion species.
13. one kind includes ion using the time of-flight mass spectrometer measurement mass spectrographic method of fragment ion, the time of-flight mass spectrometer
Source, flight path, reflector and ion detector, wherein
(a) at least two mixing time of flight spectrum are obtained using different accelerating potentials every time, the mixing time of flight spectrum bag
Signal containing the fragment ion from more than one precursor ion species, and fragment ion is in the flight path before reflector
Produce；With
(b1) mixing time of flight spectrum is compared to each other, thus to identify the letter of those fragment ions from a precursor ion species
Number；And/or
(b2) determine the flight time of fragment ion species in mixing time of flight spectrum and be used to calculate fragment ion and correlation mother
The mass-to-charge ratio of ion,
Wherein total flight path includes field-free flight path and reflector, which has with the increased electricity that slows down of quadratic power
Gesture, therefore provide system function by following equalities：
<mrow>
<mi>T</mi>
<mrow>
<mo>(</mo>
<msub>
<mi>M</mi>
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</msub>
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</mrow>
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</mrow>
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<msub>
<mi>c</mi>
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</msub>
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<msqrt>
<mfrac>
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<mi>M</mi>
<mi>p</mi>
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<mn>2</mn>
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</msub>
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</msqrt>
<mo>,</mo>
</mrow>
Wherein, MpAnd qMIt is the quality and electric charge of parent ion respectively；mp,dAnd qmIt is the quality and electric charge of fragment ion respectively；UBIt is
The accelerating potential of accelerating region before field-free flight path；UCIt is the decelerating voltage at reflector；c1And c2It is constant；And T
(Mp, mP, d) be fragment ion flight time.
14. method according to claim 13, wherein, gather and compare two mixing flight time with different accelerating potentials
Spectrum, will have in the second mixing time of flight spectrum of delay Δ T of identical flight time compared with the first mixing time of flight spectrum
All signals are determined as being derived from a precursor ion species.
15. method according to claim 14, wherein, in addition only wrapped using the accelerating potential of two mixing time of flight spectrum
Two time of flight spectrum of the signal containing parent ion, and will have the delay of identical flight time compared with the first mixing time of flight spectrum
Signal in the second mixing time of flight spectrum of Δ T is appointed as having delay Δ T's of identical flight time in time of flight spectrum
Precursor ion species.
16. method according to claim 13, wherein two mixing time of flight spectrum are obtained using different accelerating potentials, it is described
Two mixing time of flight spectrum also include the signal of related parent ion, and wherein, are determined in the second mixing time of flight spectrum
There are those signals of delay Δ T of identical flight time compared with the first mixing time of flight spectrum, and wherein, will have most long
The signal of flight time is appointed as precursor ion species, other signals are appointed as the fragment ion from the precursor ion species.
17. method according to claim 14, wherein, by the cross-correlation between the first and second mixing time of flight spectrum come really
Surely it is derived from the flight time delay of the fragment ion of different precursor ion species.
18. method according to claim 15, wherein, by the cross-correlation between the first and second mixing time of flight spectrum come really
Surely it is derived from the flight time delay of the fragment ion of different precursor ion species.
19. method according to claim 16, wherein, by the cross-correlation between the first and second mixing time of flight spectrum come really
Surely it is derived from the flight time delay of the fragment ion of different precursor ion species.
20. according to the method for any one of claim 1 to 19, wherein, ion gun uses substance assistant laser desorpted ionized
(MALDI)。
21. according to the method for any one of claim 1 to 19, wherein, by decomposing metastable state parent ion before reflector
Flight path in form fragment ion and/or fragment ion produced by parent ion in broken pond.
22. according to the method for any one of claim 1 to 19, wherein, after reflector, ion passes through accelerating region or second
Flight path, the two is all shorter than the flight path before reflector, and ion is then detected in ion detector.
23. according to the method for any one of claim 1 to 19, wherein, reflector has the potential point of Cassini ion traps
Cloth, in order to decouple the vibration of ion in machine and transverse direction.
24. according to the method for any one of claim 1 to 19, wherein, from the selection of large number of ionic species it is several it is female from
Subcategory.
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